Silver Nanoparticles in the In Vitro Aseptic Establishment of Poinsettia (Euphorbia Pulcherrima Willd. Ex Klotzsch) Var. Belén

Silver Nanoparticles in the In Vitro Aseptic Establishment of Poinsettia (Euphorbia Pulcherrima Willd. Ex Klotzsch) Var. 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Ex Klotzsch) Var. Belén Teresa de Jesús Rodríguez-Rojas, Nina Bogdanchikova, María Andrade Rodríguez, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7699181/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Feb, 2026 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 4 You are reading this latest preprint version Abstract Poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch) is the worldwide symbol of Christmas, and its demand constantly grows. The main challenge for the poinsettia in vitro propagation protocol is to maintain aseptic conditions in the culture, as the latex present in poinsettia stems significantly contributes to carbohydrates and other molecules that promote the development of endogenous phytopathogens in the culture medium after establishment. Therefore, the primary objective of this work is to assess the efficacy of AgNPs in achieving an aseptic in vitro culture using six concentrations of AgNPs (0, 100, 200, 300, 400, 500, and 600 mg L-1) added to the MS culture medium. Entirely aseptic nodal segments were obtained with the addition of 300 to 600 mg/L of AgNPs. The best shoot induction and growth promotion without compromising explant viability was obtained with the addition of 400 mg L-1 to the medium. It is essential to note that shoot induction and growth promotion were achieved without the addition of 6-benzylaminopurine (BA) or naphthalene acetic acid (NAA), which have previously been reported as the optimal conditions for poinsettia micropropagation. These results highlight the potential of AgNPs to accomplish both tasks for in vitro establishment of poinsettia cultures, eliminating bacterial and fungal contamination, and promoting conditions for shoot induction and growth. Euphorbia pulcherrima poinsettia silver nanoparticles nodal segments in vitro culture micropropagation aseptic culture Figures Figure 1 Figure 2 Figure 3 Introduction The poinsettia plant is one of the most popular ornamental plants in North America, Australia, and various European countries (Castellanos et al. 2010 ). It is the symbol of Christmas (Clarke et al. 2008 ) and is one of the most economically significant ornamental plants worldwide (Jacobo Villegas et al. 2015 ). The popularity of the plant relies on its bright, colorful bracts, which range from red to white, being fresh and unbroken for three to four months (Vilperte et al.,. The shrub is a winter-flowering species native to the states of Guerrero and Morelos in the South-central region of Mexico, growing more than 3 m in this habitat (Trejo et al. 2012 ). However, it is essential to note that the cultivation of poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch) for commercial purposes depends on varieties generated abroad, with a constant demand for new varieties of this species (Canul-Ku et al. 2018 ). In Mexico, there are nine varieties of sun poinsettia registered in the National Catalogue of Plant Varieties: “Valenciana” (red), “Juan Pablo” (pink), “Amanecer navideño” (white), and “Rehilete” and “Belén” (red) (González-García et al. 2022 ). Currently, there are approximately 300 cultivars resulting from genetic improvement and biotechnology (Trejo et al. 2018 ). Unfortunately, not enough attention has been paid to the preservation of the native Euphorbia pulcherrima . Particularly for variety Belén, genetic resources produce a limited number of commercial plants in their natural habitat, hence the need to explore and exploit the species. The poinsettia can be propagated conventionally by seeds and cuttings. However, seeds lose their viability during storage, while propagation through cuttings is seasonal. Furthermore, rooting of these cuttings takes between six and eight weeks (Danial and Ibrahim 2016 ). Moreover, as not all cuttings survive, it is worth mentioning that this method requires improved propagation techniques, which limit the available plant material for both commercial purposes and the conservation of plant genetic resources. In vitro propagation is an alternative to conventional propagation methods, as it increases the multiplication rate and generates pathogen-free material (Chávez-García et al. 2020 ). It also produces genetically homogeneous and invigorated plants, achieved by culturing tissues in an artificial medium rich in nutrients and organic compounds for multiplication under laboratory conditions (Bello-Bello and Spinoso-Castollo 2023 ). However, despite the advantages of this technique, the success of biotechnology plant propagation systems largely depends on controlling and preventing microbial contamination. This fact is particularly evident in contamination produced by endogenous species in latex-producing plants, which exploit the abundance of nutrients present in the culture medium (Cassells 1991 ; Szewczyk-Taranek et al. 2020 ). Advances in bionanotechnology provide modern alternatives for pathogen control, particularly the use of metallic nanoparticles. Among all metallic nanoparticles, silver nanoparticles (AgNPs) have shown excellent antifungal and antimicrobial properties (Alfosea-Simón et al. 2025 ). In our research group, an AgNP formulation with a specific Ag: coating agent ratio has demonstrated a broad spectrum of antimicrobial activity on vegetal systems (Vazquez-Muñoz et al. 2014 ; Andújar et al. 2020 ; Stephano-Hornedo et al. 2020 ) with minimal phytotoxic effects evaluated in the reference model, Allium cepa (Casillas-Figueroa et al. 2020 ). Additionally, this AgNPs formulation has excellent potential in plant tissue culture, for plant health, in vitro germplasm conservation, metabolite production, genetic improvement, biotechnology, and micropropagation (Bello-Bello et al. 2017 ; Pérez-Caselles et al. 2023 ; Cabrera-Ramírez et al. 2024 ; Mendoza et al. 2025 ). It is well known that the primary challenge in an in vitro propagation protocol for poinsettia is obtaining an aseptic culture, as the latex present in poinsettia stems serves as a source of endogenous phytopathogens, which can be expressed or manifested in the culture medium after establishment (Leifert and Cassells 2001 ; Perera and Trader 2010 ; Almzori et al. 2023 ). Therefore, the primary objective of this work is to evaluate the efficacy of NPsAg in achieving an aseptic in vitro culture, by assessing six concentrations of AgNPs added to the MS culture medium. Furthermore, we assess the effect of AgNPs on the length, diameter, and number of shoots generated, as well as the number of leaves on each shoot, as indicators of explant viability. Materials and Methods The research was carried out in the biotechnology laboratory of the Escuela de Estudios Superiores de Xalostoc (UAEM), Morelos, Mexico (18° 44’ 39’’ N and 98° 54’ 34’’ W, 1 294 m.a.s.l.). The climate in the region is warm semi-humid, with an average temperature of 21–24°C, low relative humidity, and average rainfall ranging between 720 and 820 mm. Silver nanoparticles Argovit-C™ is a commercial formulation of silver nanoparticles, provided by Vector-Vita Ltd. Research and Production Center, Novosibirsk, Russia. Argovit-C is a stable suspension of silver nanoparticles with a final concentration of 200 mg/L. The formulation composition corresponds to 12 mg/ml of metallic silver stabilized with 188 mg/L HP/PVP ratio 7/3 (HP = hydrolyzed protein, PVP = polyvinylpyrrolidone). These AgNPs exhibited a spherical morphology with an average diameter of 14.95 ± 10.1 nm (based on 333 particles), spanning a size distribution range of 1 to 55 nm. The UV-visible spectrum for AgNPs at 0.03 mg/ml revealed the surface plasmon resonance at 454 nm. The hydrodynamic diameter distribution of AgNPs in ultrapure water yielded two size distributions (44 and 164 nm), with a zeta potential of + 9.6 ± 0.6. The complete characterization of Argovit C was previously reported (Garcia Garcia et al. 2023 ). Plant specimens and disinfection protocol Nodal segments with an axillary bud devoid of leaves, approximately 1.0 cm, were taken from the middle part of the Belén poinsettia mother plants. In a laminar flow hood, the nodal segments were immersed in a solution containing 4g/L of Captan® (N-(trichloromethylthio) cyclohex-4-ene-1,2-dicarboximide, broad-spectrum fungicide) and 1 g/L Agri-micyn 500® (Copper 42.4% (tribasic copper sulfate) + streptomycin 1.755% + oxytetracycline 0.176%, broad-spectrum bacteriocide and fungicidal) diluted in distilled water. The nodal segments were kept under constant stirring for 30 min and subsequently rinsed three times with sterile distilled water. A 3% sodium hypochlorite (NaOCl) solution with 0.5 ml/L of detergent (escudo®) was used for surface disinfection. The samples were kept under constant stirring for 10 min and rinsed three times with sterile distilled water. Finally, the samples were immersed in 70% (v/v) ethanol for 2 min and rinsed three times with sterile distilled water. The Murashige and Skoog (MS) culture medium was supplemented with 100 mg/L of myo-inositol, 1 mg/L of thiamine HCl, 1 mg/L of pyridoxine HCl, 1 mg/L of niacin, 1 mg/L of nicotinic acid, 1 mg/L of glycine, 3% sucrose, 0.6% agar-agar, and 2.0 g/L of activated charcoal. The pH was adjusted to 5.7. The medium was dispensed in 100 mL flasks, into which 20 mL of culture medium was placed and sterilized in a Stik® MJ-54 A autoclave (Provedor de Laboratorios, S.A. de C.V., Tlajomulco de Zúñiga, Mexico) for 18 minutes at 120°C and 1.5 kg cm-2. Six treatments of silver nanoparticles (AgNPs) Argovit-C were evaluated (0, 200, 300, 400, 500, 600 mg/L), added to the MS culture medium, and subsequently sterilized. The AgNPs concentrations reported in this work are expressed in two forms: 1) metallic silver + coating agent concentration (mg/L) and 2) the metallic silver contained in the AgNP formulation (mg/L) (Table 1 ). Table 1 AgNPs concentrations used for each treatment. Concentrations correspond to metallic silver + coating agent concentration (mg/L) and the metallic silver contained in the AgNP formulation (mg/L) AgNPs treatment Metallic silver + coating agent (mg/L) Metallic silver (Ag) (mg/L) Metallic silver (mM) Control 0 0 0 Treatment 2 200 11.97 0.11 Treatment 3 300 17.96 0.16 Treatment 4 400 23.95 0.22 Treatment 5 500 29.94 0.27 Treatment 6 600 35.92 0.33 Establishment of treatments Working in a laminar flow hood, we placed three poinsettia nodal segments per flask containing culture medium for each AgNPs treatment. The flasks containing the explants were incubated in a climate chamber at 25°C, with a 16/8 h light/dark photoperiod, and a light intensity of 29 µE/m 2 s 1 . A completely randomized experimental design was used with six AgNPs treatments and 30 replicates. Each flask containing three Belén poinsettia nodal segments represents an experimental unit. After 45 days, the explants were assessed for aseptic efficacy and expressed as percentage survival (%). The number of shoots, shoot length (mm), shoot diameter (mm), and number of leaves per shoot were also evaluated. Statistical analysis The data obtained were processed using analysis of variance and a comparison of means test (Tukey, P ≤ 0.05). Statistical analysis was performed using SAS software (SAS Institute Inc 2013 ). Figures were generated with GraphPad Prism 10. Results and discussion Effect of AgNPs on asepsis of culture media The asepsis of the nodal segments of poinsettia was evaluated after 45 days of exposure to different concentrations of AgNPs. To our knowledge, this is the first time that AgNPs are used to obtain an aseptic nodal segment of poinsettia for micropropagation. The results are reported in Table 2 and Fig. 1 . The asepsis of the control group under the experimental conditions of this study was practically nonexistent; a 0.33% asepsis rate was observed. The lower assessed concentration of AgNPs produces just 20% asepsis. On the other hand, concentrations from 300 to 600 mg/L produce a 100% asepsis. The AgNPs concentration to achieve 50% asepsis is 213.8 mg/L. Sterilization of culture media by autoclaving can lead to toxic decomposition products; meanwhile, chemical sterilization using NaOCl, Ca(OCl)2, H2O2, or HgCl2 can be harmful to the explants and affect propagation efficiency (Tung et al.,. Previous works reported the chemical disinfection of the nodal segment of poinsettia, achieving asepsis rates of up to 60% using 20% NaOCl for 15 to 25 minutes (Perera and Trader 2010 ; Rangel-Estrada et al. 2017 ). On the other hand, Rangel-Estrada and co-workers reported that the best average survival rate of explants was 50%, achieved with explants aged two months and immersion time in NaOCl of 20 min (Rangel-Estrada et al. 2017 ). Table 2 Asepsis on nodal segments of poinsettia observed after exposure to different concentrations of AgNPs. AgNPs concentration in mg/L * 0 (0) 200 (11.97) 300 (17.96) 400 (23.95) 500 (29.94) 600 (35.92) Asepsis (%) 0.33 ± 0.30 c 20 ± 0.40 b 100 ± 0.37 c 100 ± 0 a 100 ± 0 a 100 ± 0 a *Concentration in parentheses corresponds to metallic silver within the silver nanoparticle formulation. Different letters in the same columns correspond to statistically significant differences. Data expressed as average value ± standard deviation. The major problem in achieving complete asepsis in the nodal segments of the poinsettia may be associated with the high microbial activity in the latex due to the abundance of organic compounds. The richer the latex in carbohydrates, the higher the microbial population (Salomez et al. 2014 ). Latex plays a significant role in plant defense due to the concentration of bioactive metabolites and defense-related enzymes, which control the growth of foreign microorganisms. Still, no damage is produced to endogenous bacteria (Salomé-Abarca et al. 2021 ). Endogenous bacteria take advantage of the opportunity provided by a nutrient-rich culture medium and the lack of competition from other microorganisms to grow rapidly, competing with the explant for nutrients and compromising its development (Leifert and Cassells 2001 ). In this work, the use of a broad-spectrum fungicide (Captan-500), a broad-spectrum bactericide (Agri-mycin 500), and NaOCl 3% in the disinfection protocol were useless in avoiding the proliferation of endogenous bacteria. The control group exhibits 99.7% contamination (Table 2 ). Conversely, the presence of AgNPs in the culture media controls the contamination in a dose-dependent manner, reaching complete elimination at 300 mg/L. The continuous presence of AgNPs in the culture media allows their interaction with the endogenous bacteria, affecting several molecular processes, including ROS overproduction, cytoskeleton compromise, and direct interaction with DNA and DNA repair enzymes, which lead to the elimination of fungi and bacteria (Guerra et al. 2020 ; Rodrigues et al. 2024 ). The effectiveness of AgNPs to produce aseptic cultures for micropropagation has been demonstrated in several vegetative systems. AgNPs with a size of < 20 nm with β-chitosan as stabilizer exhibit outstanding disinfection results of chrysanthemum plantlets using a pretreatment with AgNPs concentration from 125–500 mg/L and a few drops of Tween 80 for 5–30 minutes. Subsequently, 4–5 mg/L of AgNPs were added to the culture media to evaluate the asepsis percentage every week for four weeks. This method achieves 100% asepsis from the first week by simply adding AgNPs to culture media without autoclaving (Tung et al. 2021 ). In gladiolus apices disinfection, 91.67% of asepsis was achieved with pretreatment of 50–100 mg/L of PVP-AgNPs of 35 nm and immersion time of 10 minutes, followed by the addition of 25–200 mg/L of AgNPs to the culture medium. In this case, AgNPs were used as an auxiliary in the disinfection process. Before exposure to AgNPs, the disinfection process involves immersion in 3% NaOCl + 0.5g/l of detergent for 5 minutes (Chávez-García et al. 2020 ). For shrubby specimens, such as those of the Rosaceae family, the use of 200 mg/L AgNPs and an immersion time of 20 minutes yields the highest reduction in contamination without affecting explant survival (Shokri et al. 2015 ). Contamination control of G x N15 (Garnem), a hybrid of Prunus amygdalus (Garfi) × Prunus persica (Nemared), was achieved under micropropagation conditions using 100 mg/L of AgNPs by either immersion or addition to the culture medium (Arab et al.,. Similarly, biogenically produced AgNPs eliminate contamination of culture media used for Rumex nervosus micropropagation at a concentration of 40 mg/L (Alfarraj et al.,. On the other hand, it has been reported that the shoot tip disinfection of P. friedrichsthalianum (O. Berg) Nied. partially eliminates contamination using 50 mg/L PVP-AgNPs (Andújar et al. 2020 ). In all cases, the disinfection process includes the use of NaOCl or HgCl 2 . The above data demonstrates that the effectiveness of AgNPs relies on the nature of the AgNPs, the concentration, and the vegetal system used. In our system, AgNPs concentrations of 300 to 600 mg/L efficiently eliminate the bacterial and fungal contamination in the in vitro establishment of poinsettia cultures. Effect of AgNPs on poinsettia micropropagation The effect of different concentrations of AgNPs on poinsettia was evaluated by measuring the response in the number of shoots per explant, the length and diameter of the shoots, and the number of leaves produced per shoot. Results of the parameters mentioned above are shown in Table 3 and Fig. 2 . Table 3 Effect of AgNPs administration on shoots per explant, length and diameter of shoots, and leaves per shoot obtained in vitro from nodal segments of poinsettia. Concentration AgNPs (mg L − 1 ) * Shoots per explant (#) Shoot length (mm) Shoot diameter (mm) Leaves per shoot (#) 0 (0) 0.10 ± 0.31 c 0.76 ± 2.4 c 0.07 ± 0.21 d 0.10 ± 0.31 c 200 (11.97) 0.20 ± 0.41 c 2.82 ± 5.9 c 0.16 ± 0.36 d 0.33 ± 0.53 c 300 (17.96) 0.83 ± 0.38 b 30.63 ± 13.2 b 1.16 ± 0.42 c 2.5 ± 0.60 b 400 (23.95) 1.06 ± 0.19 a 46.04 ± 7.4 a 1.74 ± 0.28 a 4.2 ± 0.25 a 500 (29.94) 1.01 ± 0.05 a 50.29 ± 6.4 a 1.34 ± 0.11 b 2.76 ± 0.25 b 600 (35.92) 1.00 ± 0.00 ab 46.35 ± 5.9 a 1.10 ± 0.10 c 2.50 ± 0.26 b * Values in parentheses correspond to the amount of metallic silver administered. Different letters in the same columns correspond to statistically significant differences. Data expressed as average value ± standard deviation. The administration of AgNPs produces a concentration-dependent increase in the number of shoots in the nodal segments of poinsettia. The highest number of shoots was obtained with a concentration of 400 mg/L (1.06 ± 0.19 shoots per explant) and remains unchanged with the increase in AgNPs concentration (Fig. 2 a). A concentration of 300 mg/L exhibits no statistically significant difference in shoots per explant compared to the concentration of 400 mg/L (Table 3 ). The length of the shoots also shows an AgNPs concentration-dependent response from 200 to 500 mg/L. The maximum length observed was 50.29 ± 6.4 mm (Table 3 ). Conversely, although there is no statistically significant difference, with a concentration of 600 mg/L, the length of the shoot decreases to 46.35 ± 5.9 mm (Fig. 2 b). A similar trend to that described for shoot length was observed for the shoot diameter and the number of leaves per shoot; however, the values for the latter parameters decreased by 500 and 600 mg/L. The administration of AgNPs to the culture media at 400 mg/L results in a 10-fold increase in shoot production, a 66-fold increase in shoot length, a 25-fold increase in shoot diameter, and a 42-fold increase in leaves per shoot, compared with the control (Table 3 and Figs. 2 c and 2 d). It is essential to note that the longest shoots obtained with 500 and 600 mg/L of AgNPs also exhibit rolled and curly leaves with a significant decrease in the leaf area (Fig. 3 ). Leaf rolling is a typical plant response to environmental stresses, such as the overproduction of reactive oxygen species (ROS) (Kadioglu et al. 2012 ). The high concentration of AgNPs could produce the leaf rolling observed on poinsettia due to an overproduction of ROS. It has been reported that PVP-AgNPs Argovit enhances ROS production, inducing a growth-promoting response in plants at low doses (Bello-Bello et al. 2017 ; Casillas-Figueroa et al. 2020 ; Chávez-García et al. 2020 ; Mendoza et al. 2025 ). ROS concentration exhibits a dose-dependent response, producing an increase in polyphenols as a defensive response in various plant systems, including vanilla (Spinoso-Castillo et al. 2017 ), sugarcane (Bello-Bello et al. 2017 ), gerbera (Mosqueda-Frómeta et al. 2023 ), and Allium cepa (Casillas‐Figueroa. A hormetic response is observed when the molecular and enzymatic antioxidant response of plants are overwhelmed. The toxic effects of AgNPs on the mentioned plant systems are observed in the range of 50 to 250 mg/L, depending on the plant species. In this work, the hormetic response is observed with concentrations above 400 mg/L (Figs. 2 c, 2 d, and 3 ). The macroscopic effects that enable us to propose the phytotoxic impact associated with high concentrations of AgNPs are the decrease in shoot length and diameter, alongside a reduction in the number of leaves per explant (Table 3 , Fig. 2 ), and browning of the shoots (data not shown). Conversely, AgNPs concentrations of 300 and 400 mg/L enhance the length, diameter, and number of leaves per shoot without evident change in color or shape of the leaves (Table 3 , Figs. 2 and 3 ). Interestingly, shoot induction and growth promotion were achieved in this work without the addition of 6-benzylaminopurine (BA) or naphthalene acetic acid (NAA), which have previously been reported as the optimal conditions for poinsettia in vitro propagation (Almzori et al.,. Results from Table 3 show that one shoot per explant, with a length of 46 mm and 4.2 leaves per explant, were obtained with an AgNPs concentration of 400 mg/L (0.22 mM of metallic silver). Almzori reports 2.62 shoots per explant and 21.6 mm with the addition of 1 mg/L of BA. To obtain the maximum number of leaves per explant (20.41 leaves per explant), the BA concentration needs to increase to 2 mg/L (Almzori et al. 2023 ). Perera reports that BA between 0.9 and 2.7 mg/L was the most efficient cytokinin for poinsettia multiplication during micropropagation processes (Perera and Trader 2010 ). Pickens developed axillary shoots and organogenesis protocols for poinsettia using 0.36–1.5 mg/Lof BA and 0.49 mg/L of indole-3-acetic acid (IAA), producing 2.5 shoots per explant (Pickens et al. 2005 ). Additionally, five shoots per explant were obtained by adding 0.99 mg/L µM of BA and 0.08 mg/L of NAA to the culture medium (Castellanos et al.,. In summary, adding AgNPs without cytokinin or auxins to culture media yields half the number of shoots compared to using cytokinin and auxins, which are considered optimal conditions for shoot production and length. In our work, it was shown that the use of 400 mg/L of AgNPs led to an increase in shoot production and their length by 10 and 66 times, respectively. These results highlight the potential of AgNPs in promoting shoot induction and growth for the in vitro establishment of poinsettia cultures. Conclusions The results of this work demonstrate the outstanding capacity of AgNPs Argovit to eliminate bacterial and fungal contamination at concentrations of 300 and 400 mg/L, addressing one of the most significant challenges in the in vitro establishment of poinsettia micropropagation without compromising the viability of the explants. The concentration of 400 mg/L of AgNP, in addition to ensuring aseptic conditions in the culture, yields the best results in terms of shoot induction, length, and diameter, along with the highest number of leaves per shoot, without the addition of cytokinin or auxins, which have previously been reported as the optimal conditions for poinsettia micropropagation. To the best of our knowledge, this is the first report to evaluate the efficiency of AgNPs in producing aseptic conditions and stimulating growth protocols for E. pulcherrima. The results obtained in this work on the micropropagation process of (Euphorbia pulcherrima Willd. ex Klotzsch) var. Belén will contribute to a rationally designed bionanotechnology-based protocol that enables the generation of high-quality plants to meet the demand for this Christmas symbol. Declarations Authors’ ORCID Teresa de Jesús Rodríguez Rojas: https://orcid.org/0000-0003-3262-9172 Nina Bogdanchikova: https://orcid.org/0000-0003-0929-3535 María Andrade Rodríguez: https://orcid.org/0000-0003-0757-742X Alexey Pestryakov: https://orcid.org/0000-0002-9034-4733 Diana Garibo: https://orcid.org/0000-0002-6374-9014 Juan Carlos García-Ramos: https://orcid.org/0000-0001-9861-2467 Declaration of Conflicting Interests The authors report there are no conflicts of interest in this work. Author Contributions TTRR, MAR, DG, and JCGR: Conceptualization, methodology, experimental design, formal analysis, data interpretation, and writing the original draft; TTRJ, NB, and AP: funding acquisition, supervision, manuscript editing; TTRJ, NB, AP, DG, and JCGR: editing and improvement of the manuscript. The manuscript was read and approved by all authors. Funding Sources The authors declare that no funds, grants, or other support were received during the preparation of this manuscript . ACKNOWLEDGMENT The authors also thank the Russian Science Foundation, and the Tomsk region grant 22-13-20032 for help in the development of Argovit. Data Availability The data sets generated during the current study are available from the corresponding author upon reasonable request. 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Vitr Cell Dev Biol - Plant 59:507–515. https://doi.org/10.1007/s11627-023-10365-1 Perera D, Trader BW (2010) Poinsettia “prestige TM red” (Euphorbia pulcherrima) in vitro propagation. HortScience 45:1126–1128. https://doi.org/10.21273/hortsci.45.7.1126 Pérez-Caselles C, Burgos L, Sánchez-Balibrea I, et al (2023) The Effect of Silver Nanoparticle Addition on Micropropagation of Apricot Cultivars (Prunus armeniaca L.) in Semisolid and Liquid Media. Plants 12:1–17. https://doi.org/10.3390/plants12071547 Pickens KA, Cheng ZM, Trigiano RN (2005) Axillary bud proliferation and organogenesis of Euphorbia pulchurrima winter rose. Vitr Cell Dev Biol - Plant 41:770–774. https://doi.org/10.1079/IVP2005706 Rangel-Estrada SE, Canul-Ku J, Osuna-Canizalez F de J, et al (2017) In vitro regeneration of poinsettia hybrids via organogenesis. Rev Mex Ciencias Agrícolas 6:1571–1585. https://doi.org/10.29312/remexca.v6i7.551 Rodrigues AS, Batista JGS, Rodrigues MÁV, et al (2024) Advances in silver nanoparticles: a comprehensive review on their potential as antimicrobial agents and their mechanisms of action elucidated by proteomics. Front Microbiol 15:. https://doi.org/10.3389/fmicb.2024.1440065 Salomé-Abarca LF, Gođevac D, Kim MS, et al (2021) Latex Metabolome of Euphorbia Species: Geographical and Inter-Species Variation and its Proposed Role in Plant Defense against Herbivores and Pathogens. J Chem Ecol 47:564–576. https://doi.org/10.1007/s10886-021-01274-x Salomez M, Subileau M, Intapun J, et al (2014) Micro-organisms in latex and natural rubber coagula of Hevea brasiliensis and their impact on rubber composition, structure and properties. J Appl Microbiol 117:921–929. https://doi.org/10.1111/jam.12556 SAS Institute Inc (2013) Statistical Analysis Software SAS Shokri S, Babaei A, Ahmadian M, et al (2015) The effects of different concentrations of nano-silver on elimination of bacterial contaminations and phenolic exudation of rose (Rosa hybrida L.) In vitro culture. Acta Hortic 1083:391–396. https://doi.org/10.17660/ActaHortic.2015.1083.49 Spinoso-Castillo JL, Chavez-Santoscoy RA, Bogdanchikova N, et al (2017) Antimicrobial and hormetic effects of silver nanoparticles on in vitro regeneration of vanilla (Vanilla planifolia Jacks. ex Andrews) using a temporary immersion system. Plant Cell, Tissue Organ Cult 129:195–207. https://doi.org/10.1007/s11240-017-1169-8 Stephano-Hornedo JL, Torres-Gutiérrez O, Toledano-Magaña Y, et al (2020) Argovit TM silver nanoparticles to fight Huanglongbing disease in Mexican limes (: Citrus aurantifolia Swingle). RSC Adv 10:6146–6155. https://doi.org/10.1039/c9ra09018e Szewczyk-Taranek B, Jaglarz A, Pałka P, et al (2020) Identification and control of endophytic bacteria during in vitro cultures of Staphylea pinnata L. Folia Hortic 32:47–55. https://doi.org/10.2478/fhort-2020-0005 Trejo L, Arroyo TPF, Olsen KM, et al (2012) Poinsettia’s wild ancestor in the mexican dry tropics: Historical, genetic, and environmental evidence. Am J Bot 99:1146–1157. https://doi.org/10.3732/ajb.1200072 Trejo L, Rosell JA, Olson ME (2018) Nearly 200 years of sustained selection have not overcome the leaf area–stem size relationship in the poinsettia. Evol Appl 11:1401–1411. https://doi.org/10.1111/eva.12634 Tung HT, Bao HG, Cuong DM, et al (2021) Silver nanoparticles as the sterilant in large-scale micropropagation of chrysanthemum. Vitr Cell Dev Biol - Plant 57:897–906. https://doi.org/10.1007/s11627-021-10163-7 Vazquez-Muñoz R, Avalos-Borja M, Castro-Longoria E (2014) Ultrastructural analysis of candida albicans when exposed to silver nanoparticles. PLoS One 9:1–10. https://doi.org/10.1371/journal.pone.0108876 Vilperte V, Lucaciu CR, Halbwirth H, et al (2019) Hybrid de novo transcriptome assembly of poinsettia (Euphorbia pulcherrima Willd. Ex Klotsch) bracts. BMC Genomics 20:1–19. https://doi.org/10.1186/s12864-019-6247-3 Cite Share Download PDF Status: Published Journal Publication published 26 Feb, 2026 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted Reviewers agreed at journal 17 Oct, 2025 Reviewers invited by journal 02 Oct, 2025 Editor assigned by journal 27 Sep, 2025 First submitted to journal 25 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7699181","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":524015063,"identity":"d8313519-a3c2-4bbe-9623-9c6eecb72128","order_by":0,"name":"Teresa de Jesús Rodríguez-Rojas","email":"","orcid":"","institution":"Universidad Autonoma del Estado de Morelos","correspondingAuthor":false,"prefix":"","firstName":"Teresa","middleName":"de Jesús","lastName":"Rodríguez-Rojas","suffix":""},{"id":524015064,"identity":"5e5bb45f-c431-40b9-b14f-cc473046c5f2","order_by":1,"name":"Nina 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18:05:17","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":104807,"visible":true,"origin":"","legend":"","description":"","filename":"PCTOD25006950structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7699181/v1/f0d8330535ea507b717b98a4.xml"},{"id":93622564,"identity":"360010a8-7b3c-44c1-836f-daf5b22e32f7","added_by":"auto","created_at":"2025-10-15 18:13:17","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":112750,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7699181/v1/1b59c27d67248180c275ef22.html"},{"id":93621589,"identity":"3cfa0551-1ece-4bcd-a430-eca6a32cec25","added_by":"auto","created_at":"2025-10-15 18:05:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":20527,"visible":true,"origin":"","legend":"\u003cp\u003eAsepsis (%) on poinsettia nodal segments exposed to different concentrations of AgNPs after 45 days of exposure. Values are expressed as the average ± standard deviation (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7699181/v1/c6295366055bd17dc7a51431.jpg"},{"id":93622561,"identity":"9b7c27e8-897b-434c-8616-4c92933ebc84","added_by":"auto","created_at":"2025-10-15 18:13:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":43257,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different concentrations of AgNPs (mg/L) on a) shoots per explant, b) shoot length, c) shoot diameter, and d) leaves per shoot obtained in vitro from nodal segments of poinsettia.Values are expressed as the average ± standard deviation (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7699181/v1/85996ced19f3ec168994ea1e.jpg"},{"id":93621594,"identity":"1979d962-91b5-4411-8816-fce21b37f279","added_by":"auto","created_at":"2025-10-15 18:05:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":25508,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of poinsettia nodal segments development after 45 days of exposure to different concentrations of AgNPs. The concentration is indicated below each nodal segment. The black bar on the left represents 1 mm in length.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7699181/v1/938a3b654502cdf38700a930.jpg"},{"id":103766778,"identity":"5a61a4bb-0031-430b-b2f0-22382852b3cf","added_by":"auto","created_at":"2026-03-02 16:16:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":819800,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7699181/v1/eeaf1fd0-c8be-457f-82d1-cfd0d21bb73a.pdf"}],"financialInterests":"","formattedTitle":"Silver Nanoparticles in the In Vitro Aseptic Establishment of Poinsettia (Euphorbia Pulcherrima Willd. Ex Klotzsch) Var. Belén","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe poinsettia plant is one of the most popular ornamental plants in North America, Australia, and various European countries (Castellanos et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). It is the symbol of Christmas (Clarke et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and is one of the most economically significant ornamental plants worldwide (Jacobo Villegas et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The popularity of the plant relies on its bright, colorful bracts, which range from red to white, being fresh and unbroken for three to four months (Vilperte et al.,.\u003c/p\u003e\u003cp\u003eThe shrub is a winter-flowering species native to the states of Guerrero and Morelos in the South-central region of Mexico, growing more than 3 m in this habitat (Trejo et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, it is essential to note that the cultivation of poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch) for commercial purposes depends on varieties generated abroad, with a constant demand for new varieties of this species (Canul-Ku et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In Mexico, there are nine varieties of sun poinsettia registered in the National Catalogue of Plant Varieties: \u0026ldquo;Valenciana\u0026rdquo; (red), \u0026ldquo;Juan Pablo\u0026rdquo; (pink), \u0026ldquo;Amanecer navide\u0026ntilde;o\u0026rdquo; (white), and \u0026ldquo;Rehilete\u0026rdquo; and \u0026ldquo;Bel\u0026eacute;n\u0026rdquo; (red) (Gonz\u0026aacute;lez-Garc\u0026iacute;a et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Currently, there are approximately 300 cultivars resulting from genetic improvement and biotechnology (Trejo et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Unfortunately, not enough attention has been paid to the preservation of the native \u003cem\u003eEuphorbia pulcherrima\u003c/em\u003e. Particularly for variety Bel\u0026eacute;n, genetic resources produce a limited number of commercial plants in their natural habitat, hence the need to explore and exploit the species.\u003c/p\u003e\u003cp\u003eThe poinsettia can be propagated conventionally by seeds and cuttings. However, seeds lose their viability during storage, while propagation through cuttings is seasonal. Furthermore, rooting of these cuttings takes between six and eight weeks (Danial and Ibrahim \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, as not all cuttings survive, it is worth mentioning that this method requires improved propagation techniques, which limit the available plant material for both commercial purposes and the conservation of plant genetic resources.\u003c/p\u003e\u003cp\u003eIn vitro propagation is an alternative to conventional propagation methods, as it increases the multiplication rate and generates pathogen-free material (Ch\u0026aacute;vez-Garc\u0026iacute;a et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It also produces genetically homogeneous and invigorated plants, achieved by culturing tissues in an artificial medium rich in nutrients and organic compounds for multiplication under laboratory conditions (Bello-Bello and Spinoso-Castollo \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, despite the advantages of this technique, the success of biotechnology plant propagation systems largely depends on controlling and preventing microbial contamination. This fact is particularly evident in contamination produced by endogenous species in latex-producing plants, which exploit the abundance of nutrients present in the culture medium (Cassells \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Szewczyk-Taranek et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAdvances in bionanotechnology provide modern alternatives for pathogen control, particularly the use of metallic nanoparticles. Among all metallic nanoparticles, silver nanoparticles (AgNPs) have shown excellent antifungal and antimicrobial properties (Alfosea-Sim\u0026oacute;n et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In our research group, an AgNP formulation with a specific Ag: coating agent ratio has demonstrated a broad spectrum of antimicrobial activity on vegetal systems (Vazquez-Mu\u0026ntilde;oz et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; And\u0026uacute;jar et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Stephano-Hornedo et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) with minimal phytotoxic effects evaluated in the reference model, \u003cem\u003eAllium cepa\u003c/em\u003e (Casillas-Figueroa et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, this AgNPs formulation has excellent potential in plant tissue culture, for plant health, \u003cem\u003ein vitro\u003c/em\u003e germplasm conservation, metabolite production, genetic improvement, biotechnology, and micropropagation (Bello-Bello et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; P\u0026eacute;rez-Caselles et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Cabrera-Ram\u0026iacute;rez et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Mendoza et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIt is well known that the primary challenge in an \u003cem\u003ein vitro\u003c/em\u003e propagation protocol for poinsettia is obtaining an aseptic culture, as the latex present in poinsettia stems serves as a source of endogenous phytopathogens, which can be expressed or manifested in the culture medium after establishment (Leifert and Cassells \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Perera and Trader \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Almzori et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, the primary objective of this work is to evaluate the efficacy of NPsAg in achieving an aseptic in vitro culture, by assessing six concentrations of AgNPs added to the MS culture medium. Furthermore, we assess the effect of AgNPs on the length, diameter, and number of shoots generated, as well as the number of leaves on each shoot, as indicators of explant viability.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThe research was carried out in the biotechnology laboratory of the Escuela de Estudios Superiores de Xalostoc (UAEM), Morelos, Mexico (18\u0026deg; 44\u0026rsquo; 39\u0026rsquo;\u0026rsquo; N and 98\u0026deg; 54\u0026rsquo; 34\u0026rsquo;\u0026rsquo; W, 1 294 m.a.s.l.). The climate in the region is warm semi-humid, with an average temperature of 21\u0026ndash;24\u0026deg;C, low relative humidity, and average rainfall ranging between 720 and 820 mm.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSilver nanoparticles\u003c/h2\u003e\u003cp\u003eArgovit-C\u0026trade; is a commercial formulation of silver nanoparticles, provided by Vector-Vita Ltd. Research and Production Center, Novosibirsk, Russia. Argovit-C is a stable suspension of silver nanoparticles with a final concentration of 200 mg/L. The formulation composition corresponds to 12 mg/ml of metallic silver stabilized with 188 mg/L HP/PVP ratio 7/3 (HP\u0026thinsp;=\u0026thinsp;hydrolyzed protein, PVP\u0026thinsp;=\u0026thinsp;polyvinylpyrrolidone). These AgNPs exhibited a spherical morphology with an average diameter of 14.95\u0026thinsp;\u0026plusmn;\u0026thinsp;10.1 nm (based on 333 particles), spanning a size distribution range of 1 to 55 nm. The UV-visible spectrum for AgNPs at 0.03 mg/ml revealed the surface plasmon resonance at 454 nm. The hydrodynamic diameter distribution of AgNPs in ultrapure water yielded two size distributions (44 and 164 nm), with a zeta potential of +\u0026thinsp;9.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6. The complete characterization of Argovit C was previously reported (Garcia Garcia et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePlant specimens and disinfection protocol\u003c/h3\u003e\n\u003cp\u003eNodal segments with an axillary bud devoid of leaves, approximately 1.0 cm, were taken from the middle part of the Bel\u0026eacute;n poinsettia mother plants. In a laminar flow hood, the nodal segments were immersed in a solution containing 4g/L of Captan\u0026reg; (N-(trichloromethylthio) cyclohex-4-ene-1,2-dicarboximide, broad-spectrum fungicide) and 1 g/L Agri-micyn 500\u0026reg; (Copper 42.4% (tribasic copper sulfate)\u0026thinsp;+\u0026thinsp;streptomycin 1.755% + oxytetracycline 0.176%, broad-spectrum bacteriocide and fungicidal) diluted in distilled water. The nodal segments were kept under constant stirring for 30 min and subsequently rinsed three times with sterile distilled water. A 3% sodium hypochlorite (NaOCl) solution with 0.5 ml/L of detergent (escudo\u0026reg;) was used for surface disinfection. The samples were kept under constant stirring for 10 min and rinsed three times with sterile distilled water. Finally, the samples were immersed in 70% (v/v) ethanol for 2 min and rinsed three times with sterile distilled water.\u003c/p\u003e\u003cp\u003eThe Murashige and Skoog (MS) culture medium was supplemented with 100 mg/L of myo-inositol, 1 mg/L of thiamine HCl, 1 mg/L of pyridoxine HCl, 1 mg/L of niacin, 1 mg/L of nicotinic acid, 1 mg/L of glycine, 3% sucrose, 0.6% agar-agar, and 2.0 g/L of activated charcoal. The pH was adjusted to 5.7.\u003c/p\u003e\u003cp\u003eThe medium was dispensed in 100 mL flasks, into which 20 mL of culture medium was placed and sterilized in a Stik\u0026reg; MJ-54 A autoclave (Provedor de Laboratorios, S.A. de C.V., Tlajomulco de Z\u0026uacute;\u0026ntilde;iga, Mexico) for 18 minutes at 120\u0026deg;C and 1.5 kg cm-2.\u003c/p\u003e\u003cp\u003eSix treatments of silver nanoparticles (AgNPs) Argovit-C were evaluated (0, 200, 300, 400, 500, 600 mg/L), added to the MS culture medium, and subsequently sterilized. The AgNPs concentrations reported in this work are expressed in two forms: 1) metallic silver\u0026thinsp;+\u0026thinsp;coating agent concentration (mg/L) and 2) the metallic silver contained in the AgNP formulation (mg/L) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAgNPs concentrations used for each treatment. Concentrations correspond to metallic silver\u0026thinsp;+\u0026thinsp;coating agent concentration (mg/L) and the metallic silver contained in the AgNP formulation (mg/L)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAgNPs treatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMetallic silver\u0026thinsp;+\u0026thinsp;coating agent (mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMetallic silver (Ag) (mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMetallic silver (mM)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e300\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e23.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment 6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e600\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eEstablishment of treatments\u003c/h3\u003e\n\u003cp\u003eWorking in a laminar flow hood, we placed three poinsettia nodal segments per flask containing culture medium for each AgNPs treatment. The flasks containing the explants were incubated in a climate chamber at 25\u0026deg;C, with a 16/8 h light/dark photoperiod, and a light intensity of 29 \u0026micro;E/m\u003csup\u003e2\u003c/sup\u003es\u003csup\u003e1\u003c/sup\u003e. A completely randomized experimental design was used with six AgNPs treatments and 30 replicates. Each flask containing three Bel\u0026eacute;n poinsettia nodal segments represents an experimental unit. After 45 days, the explants were assessed for aseptic efficacy and expressed as percentage survival (%). The number of shoots, shoot length (mm), shoot diameter (mm), and number of leaves per shoot were also evaluated.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe data obtained were processed using analysis of variance and a comparison of means test (Tukey, P\u0026thinsp;\u0026le;\u0026thinsp;0.05). Statistical analysis was performed using SAS software (SAS Institute Inc \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Figures were generated with GraphPad Prism 10.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eEffect of AgNPs on asepsis of culture media\u003c/h2\u003e\u003cp\u003eThe asepsis of the nodal segments of poinsettia was evaluated after 45 days of exposure to different concentrations of AgNPs. To our knowledge, this is the first time that AgNPs are used to obtain an aseptic nodal segment of poinsettia for micropropagation. The results are reported in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The asepsis of the control group under the experimental conditions of this study was practically nonexistent; a 0.33% asepsis rate was observed. The lower assessed concentration of AgNPs produces just 20% asepsis. On the other hand, concentrations from 300 to 600 mg/L produce a 100% asepsis. The AgNPs concentration to achieve 50% asepsis is 213.8 mg/L.\u003c/p\u003e\u003cp\u003eSterilization of culture media by autoclaving can lead to toxic decomposition products; meanwhile, chemical sterilization using NaOCl, Ca(OCl)2, H2O2, or HgCl2 can be harmful to the explants and affect propagation efficiency (Tung et al.,. Previous works reported the chemical disinfection of the nodal segment of poinsettia, achieving asepsis rates of up to 60% using 20% NaOCl for 15 to 25 minutes (Perera and Trader \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Rangel-Estrada et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). On the other hand, Rangel-Estrada and co-workers reported that the best average survival rate of explants was 50%, achieved with explants aged two months and immersion time in NaOCl of 20 min (Rangel-Estrada et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAsepsis on nodal segments of poinsettia observed after exposure to different concentrations of AgNPs.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAgNPs concentration in mg/L\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003cp\u003e(0)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e200\u003c/p\u003e\u003cp\u003e(11.97)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e300\u003c/p\u003e\u003cp\u003e(17.96)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e400\u003c/p\u003e\u003cp\u003e(23.95)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e500\u003c/p\u003e\u003cp\u003e(29.94)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e600\u003c/p\u003e\u003cp\u003e(35.92)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAsepsis (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e*Concentration in parentheses corresponds to metallic silver within the silver nanoparticle formulation. Different letters in the same columns correspond to statistically significant differences. Data expressed as average value\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe major problem in achieving complete asepsis in the nodal segments of the poinsettia may be associated with the high microbial activity in the latex due to the abundance of organic compounds. The richer the latex in carbohydrates, the higher the microbial population (Salomez et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Latex plays a significant role in plant defense due to the concentration of bioactive metabolites and defense-related enzymes, which control the growth of foreign microorganisms. Still, no damage is produced to endogenous bacteria (Salom\u0026eacute;-Abarca et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Endogenous bacteria take advantage of the opportunity provided by a nutrient-rich culture medium and the lack of competition from other microorganisms to grow rapidly, competing with the explant for nutrients and compromising its development (Leifert and Cassells \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this work, the use of a broad-spectrum fungicide (Captan-500), a broad-spectrum bactericide (Agri-mycin 500), and NaOCl 3% in the disinfection protocol were useless in avoiding the proliferation of endogenous bacteria. The control group exhibits 99.7% contamination (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Conversely, the presence of AgNPs in the culture media controls the contamination in a dose-dependent manner, reaching complete elimination at 300 mg/L. The continuous presence of AgNPs in the culture media allows their interaction with the endogenous bacteria, affecting several molecular processes, including ROS overproduction, cytoskeleton compromise, and direct interaction with DNA and DNA repair enzymes, which lead to the elimination of fungi and bacteria (Guerra et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rodrigues et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe effectiveness of AgNPs to produce aseptic cultures for micropropagation has been demonstrated in several vegetative systems. AgNPs with a size of \u0026lt;\u0026thinsp;20 nm with β-chitosan as stabilizer exhibit outstanding disinfection results of chrysanthemum plantlets using a pretreatment with AgNPs concentration from 125\u0026ndash;500 mg/L and a few drops of Tween 80 for 5\u0026ndash;30 minutes. Subsequently, 4\u0026ndash;5 mg/L of AgNPs were added to the culture media to evaluate the asepsis percentage every week for four weeks. This method achieves 100% asepsis from the first week by simply adding AgNPs to culture media without autoclaving (Tung et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn gladiolus apices disinfection, 91.67% of asepsis was achieved with pretreatment of 50\u0026ndash;100 mg/L of PVP-AgNPs of 35 nm and immersion time of 10 minutes, followed by the addition of 25\u0026ndash;200 mg/L of AgNPs to the culture medium. In this case, AgNPs were used as an auxiliary in the disinfection process. Before exposure to AgNPs, the disinfection process involves immersion in 3% NaOCl\u0026thinsp;+\u0026thinsp;0.5g/l of detergent for 5 minutes (Ch\u0026aacute;vez-Garc\u0026iacute;a et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor shrubby specimens, such as those of the Rosaceae family, the use of 200 mg/L AgNPs and an immersion time of 20 minutes yields the highest reduction in contamination without affecting explant survival (Shokri et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Contamination control of G x N15 (Garnem), a hybrid of Prunus amygdalus (Garfi) \u0026times; Prunus persica (Nemared), was achieved under micropropagation conditions using 100 mg/L of AgNPs by either immersion or addition to the culture medium (Arab et al.,. Similarly, biogenically produced AgNPs eliminate contamination of culture media used for \u003cem\u003eRumex nervosus\u003c/em\u003e micropropagation at a concentration of 40 mg/L (Alfarraj et al.,. On the other hand, it has been reported that the shoot tip disinfection of P. friedrichsthalianum (O. Berg) Nied. partially eliminates contamination using 50 mg/L PVP-AgNPs (And\u0026uacute;jar et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In all cases, the disinfection process includes the use of NaOCl or HgCl\u003csub\u003e2\u003c/sub\u003e. The above data demonstrates that the effectiveness of AgNPs relies on the nature of the AgNPs, the concentration, and the vegetal system used.\u003c/p\u003e\u003cp\u003eIn our system, AgNPs concentrations of 300 to 600 mg/L efficiently eliminate the bacterial and fungal contamination in the in vitro establishment of poinsettia cultures.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEffect of AgNPs on poinsettia micropropagation\u003c/h3\u003e\n\u003cp\u003eThe effect of different concentrations of AgNPs on poinsettia was evaluated by measuring the response in the number of shoots per explant, the length and diameter of the shoots, and the number of leaves produced per shoot. Results of the parameters mentioned above are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of AgNPs administration on shoots per explant, length and diameter of shoots, and leaves per shoot obtained \u003cem\u003ein vitro\u003c/em\u003e from nodal segments of poinsettia.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConcentration AgNPs (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) *\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eShoots per explant (#)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eShoot length (mm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eShoot diameter (mm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLeaves per shoot (#)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e200 (11.97)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.82\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e300 (17.96)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30.63\u0026thinsp;\u0026plusmn;\u0026thinsp;13.2 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e400 (23.95)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46.04\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e500 (29.94)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50.29\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e600 (35.92)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46.35\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e* Values in parentheses correspond to the amount of metallic silver administered. Different letters in the same columns correspond to statistically significant differences. Data expressed as average value\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe administration of AgNPs produces a concentration-dependent increase in the number of shoots in the nodal segments of poinsettia. The highest number of shoots was obtained with a concentration of 400 mg/L (1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 shoots per explant) and remains unchanged with the increase in AgNPs concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). A concentration of 300 mg/L exhibits no statistically significant difference in shoots per explant compared to the concentration of 400 mg/L (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The length of the shoots also shows an AgNPs concentration-dependent response from 200 to 500 mg/L. The maximum length observed was 50.29\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4 mm (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Conversely, although there is no statistically significant difference, with a concentration of 600 mg/L, the length of the shoot decreases to 46.35\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). A similar trend to that described for shoot length was observed for the shoot diameter and the number of leaves per shoot; however, the values for the latter parameters decreased by 500 and 600 mg/L. The administration of AgNPs to the culture media at 400 mg/L results in a 10-fold increase in shoot production, a 66-fold increase in shoot length, a 25-fold increase in shoot diameter, and a 42-fold increase in leaves per shoot, compared with the control (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003eIt is essential to note that the longest shoots obtained with 500 and 600 mg/L of AgNPs also exhibit rolled and curly leaves with a significant decrease in the leaf area (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eLeaf rolling is a typical plant response to environmental stresses, such as the overproduction of reactive oxygen species (ROS) (Kadioglu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The high concentration of AgNPs could produce the leaf rolling observed on poinsettia due to an overproduction of ROS. It has been reported that PVP-AgNPs Argovit enhances ROS production, inducing a growth-promoting response in plants at low doses (Bello-Bello et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Casillas-Figueroa et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ch\u0026aacute;vez-Garc\u0026iacute;a et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mendoza et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). ROS concentration exhibits a dose-dependent response, producing an increase in polyphenols as a defensive response in various plant systems, including vanilla (Spinoso-Castillo et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), sugarcane (Bello-Bello et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), gerbera (Mosqueda-Fr\u0026oacute;meta et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and Allium cepa (Casillas‐Figueroa. A hormetic response is observed when the molecular and enzymatic antioxidant response of plants are overwhelmed. The toxic effects of AgNPs on the mentioned plant systems are observed in the range of 50 to 250 mg/L, depending on the plant species.\u003c/p\u003e\u003cp\u003eIn this work, the hormetic response is observed with concentrations above 400 mg/L (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The macroscopic effects that enable us to propose the phytotoxic impact associated with high concentrations of AgNPs are the decrease in shoot length and diameter, alongside a reduction in the number of leaves per explant (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and browning of the shoots (data not shown). Conversely, AgNPs concentrations of 300 and 400 mg/L enhance the length, diameter, and number of leaves per shoot without evident change in color or shape of the leaves (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eInterestingly, shoot induction and growth promotion were achieved in this work without the addition of 6-benzylaminopurine (BA) or naphthalene acetic acid (NAA), which have previously been reported as the optimal conditions for poinsettia in vitro propagation (Almzori et al.,. Results from Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e show that one shoot per explant, with a length of 46 mm and 4.2 leaves per explant, were obtained with an AgNPs concentration of 400 mg/L (0.22 mM of metallic silver). Almzori reports 2.62 shoots per explant and 21.6 mm with the addition of 1 mg/L of BA. To obtain the maximum number of leaves per explant (20.41 leaves per explant), the BA concentration needs to increase to 2 mg/L (Almzori et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Perera reports that BA between 0.9 and 2.7 mg/L was the most efficient cytokinin for poinsettia multiplication during micropropagation processes (Perera and Trader \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Pickens developed axillary shoots and organogenesis protocols for poinsettia using 0.36\u0026ndash;1.5 mg/Lof BA and 0.49 mg/L of indole-3-acetic acid (IAA), producing 2.5 shoots per explant (Pickens et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Additionally, five shoots per explant were obtained by adding 0.99 mg/L \u0026micro;M of BA and 0.08 mg/L of NAA to the culture medium (Castellanos et al.,. In summary, adding AgNPs without cytokinin or auxins to culture media yields half the number of shoots compared to using cytokinin and auxins, which are considered optimal conditions for shoot production and length. In our work, it was shown that the use of 400 mg/L of AgNPs led to an increase in shoot production and their length by 10 and 66 times, respectively. These results highlight the potential of AgNPs in promoting shoot induction and growth for the \u003cem\u003ein vitro\u003c/em\u003e establishment of poinsettia cultures.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe results of this work demonstrate the outstanding capacity of AgNPs Argovit to eliminate bacterial and fungal contamination at concentrations of 300 and 400 mg/L, addressing one of the most significant challenges in the \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eestablishment of poinsettia micropropagation without compromising the viability of the explants. The concentration of 400 mg/L of AgNP, in addition to ensuring aseptic conditions in the culture, yields the best results in terms of shoot induction, length, and diameter, along with the highest number of leaves per shoot, without the addition of cytokinin or auxins, which have previously been reported as the optimal conditions for poinsettia micropropagation. To the best of our knowledge, this is the first report to evaluate the efficiency of AgNPs in producing aseptic conditions and stimulating growth protocols for E. pulcherrima. The results obtained in this work on the micropropagation process of (Euphorbia pulcherrima Willd. ex Klotzsch) var. Bel\u0026eacute;n will contribute to a rationally designed bionanotechnology-based protocol that enables the generation of high-quality plants to meet the demand for this Christmas symbol.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; ORCID\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTeresa de Jes\u0026uacute;s Rodr\u0026iacute;guez Rojas: https://orcid.org/0000-0003-3262-9172\u003c/p\u003e\n\u003cp\u003eNina Bogdanchikova: https://orcid.org/0000-0003-0929-3535 \u003c/p\u003e\n\u003cp\u003eMar\u0026iacute;a Andrade Rodr\u0026iacute;guez: https://orcid.org/0000-0003-0757-742X\u003c/p\u003e\n\u003cp\u003eAlexey Pestryakov: https://orcid.org/0000-0002-9034-4733\u003c/p\u003e\n\u003cp\u003eDiana Garibo: https://orcid.org/0000-0002-6374-9014\u003c/p\u003e\n\u003cp\u003eJuan Carlos Garc\u0026iacute;a-Ramos: https://orcid.org/0000-0001-9861-2467 \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Conflicting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report there are no conflicts of interest in this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTTRR, MAR, DG, and JCGR: Conceptualization, methodology, experimental design, formal analysis, data interpretation, and writing the original draft; TTRJ, NB, and AP: funding acquisition, supervision, manuscript editing; TTRJ, NB, AP, DG, and JCGR: editing and improvement of the manuscript. The manuscript was read and approved by all authors. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors also thank the Russian Science Foundation, and the Tomsk region grant 22-13-20032 for help in the development of Argovit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability \u003c/strong\u003eThe data sets generated during the current study are available from the corresponding author upon reasonable request. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration\u003c/strong\u003e Not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlfarraj NS, Tarroum M, Al-Qurainy F, et al (2023) Biosynthesis of Silver Nanoparticles and Exploring Their Potential of Reducing the Contamination of the In Vitro Culture Media and Inducing the Callus Growth of Rumex nervosus Explants. 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Rev Mex Ciencias Agr\u0026iacute;colas 13:433\u0026ndash;442. https://doi.org/10.29312/remexca.v13i3.2831\u003c/li\u003e\n\u003cli\u003eGuerra JD, Sandoval G, Patron A, et al (2020) Selective antifungal activity of silver nanoparticles: A comparative study between Candida tropicalis and Saccharomyces boulardii. Colloids Interface Sci Commun 37:100280. https://doi.org/10.1016/j.colcom.2020.100280\u003c/li\u003e\n\u003cli\u003eJacobo Villegas O, Valdovinos Ponce G, Ram\u0026iacute;rez Rojas S, Hern\u0026aacute;ndez Ju\u0026aacute;rez C (2015) B\u0026uacute;squeda de fuentes de resistencia al Poinsettia mosaic virus en plantas silvestres de nochebuena. Search sources Resist to Poinsettia mosaic virus wild poinsettia plants 33:219\u0026ndash;231\u003c/li\u003e\n\u003cli\u003eKadioglu A, Terzi R, Saruhan N, Saglam A (2012) Current advances in the investigation of leaf rolling caused by biotic and abiotic stress factors. 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Vitr Cell Dev Biol - Plant 41:770\u0026ndash;774. https://doi.org/10.1079/IVP2005706\u003c/li\u003e\n\u003cli\u003eRangel-Estrada SE, Canul-Ku J, Osuna-Canizalez F de J, et al (2017) In vitro regeneration of poinsettia hybrids via organogenesis. Rev Mex Ciencias Agr\u0026iacute;colas 6:1571\u0026ndash;1585. https://doi.org/10.29312/remexca.v6i7.551\u003c/li\u003e\n\u003cli\u003eRodrigues AS, Batista JGS, Rodrigues M\u0026Aacute;V, et al (2024) Advances in silver nanoparticles: a comprehensive review on their potential as antimicrobial agents and their mechanisms of action elucidated by proteomics. Front Microbiol 15:. https://doi.org/10.3389/fmicb.2024.1440065\u003c/li\u003e\n\u003cli\u003eSalom\u0026eacute;-Abarca LF, Gođevac D, Kim MS, et al (2021) Latex Metabolome of Euphorbia Species: Geographical and Inter-Species Variation and its Proposed Role in Plant Defense against Herbivores and Pathogens. 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Vitr Cell Dev Biol - Plant 57:897\u0026ndash;906. https://doi.org/10.1007/s11627-021-10163-7\u003c/li\u003e\n\u003cli\u003eVazquez-Mu\u0026ntilde;oz R, Avalos-Borja M, Castro-Longoria E (2014) Ultrastructural analysis of candida albicans when exposed to silver nanoparticles. PLoS One 9:1\u0026ndash;10. https://doi.org/10.1371/journal.pone.0108876\u003c/li\u003e\n\u003cli\u003eVilperte V, Lucaciu CR, Halbwirth H, et al (2019) Hybrid de novo transcriptome assembly of poinsettia (Euphorbia pulcherrima Willd. Ex Klotsch) bracts. BMC Genomics 20:1\u0026ndash;19. https://doi.org/10.1186/s12864-019-6247-3\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Euphorbia pulcherrima, poinsettia, silver nanoparticles, nodal segments, in vitro culture, micropropagation, aseptic culture","lastPublishedDoi":"10.21203/rs.3.rs-7699181/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7699181/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePoinsettia (Euphorbia pulcherrima Willd. ex Klotzsch) is the worldwide symbol of Christmas, and its demand constantly grows. The main challenge for the poinsettia \u003cem\u003ein vitro\u003c/em\u003e propagation protocol is to maintain aseptic conditions in the culture, as the latex present in poinsettia stems significantly contributes to carbohydrates and other molecules that promote the development of endogenous phytopathogens in the culture medium after establishment. Therefore, the primary objective of this work is to assess the efficacy of AgNPs in achieving an aseptic \u003cem\u003ein vitro\u003c/em\u003e culture using six concentrations of AgNPs (0, 100, 200, 300, 400, 500, and 600 mg L-1) added to the MS culture medium. Entirely aseptic nodal segments were obtained with the addition of 300 to 600 mg/L of AgNPs. The best shoot induction and growth promotion without compromising explant viability was obtained with the addition of 400 mg L-1 to the medium. It is essential to note that shoot induction and growth promotion were achieved without the addition of 6-benzylaminopurine (BA) or naphthalene acetic acid (NAA), which have previously been reported as the optimal conditions for poinsettia micropropagation. These results highlight the potential of AgNPs to accomplish both tasks for \u003cem\u003ein vitro\u003c/em\u003e establishment of poinsettia cultures, eliminating bacterial and fungal contamination, and promoting conditions for shoot induction and growth.\u003c/p\u003e","manuscriptTitle":"Silver Nanoparticles in the In Vitro Aseptic Establishment of Poinsettia (Euphorbia Pulcherrima Willd. Ex Klotzsch) Var. 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